Integration and Services

Day-to-day bioprocess development is a special challenge. Here, we give practical tips at hand, starting with a discussion about the influence of leachables and extractables on product safety and how to mitigate these issues. Also, we give an overview on impeller types and how they affect mixing in a stirred-tank bioreactor.

Finding the needle in the haystack: Leachables and extractables in bioprocessing

The influence of leachables and extractables (L&E) on drug quality and safety is one of the hot topics in current biopharmaceutical processing. The L&E question, especially regarding packaging, has been discussed for years. However, with the increased use of single-use bioreactor equipment, the L&E debate touches on upstream bioprocessing as well.

L&E – What are we talking about?

Plastics are used throughout the supply chain, production, and packaging of drugs. Examples include single-use bioreactors, storage containers, disposable purification equipment, and primary packaging systems. The concern is that chemicals, organic or inorganic, could leach from the plastics into solution in liquid pharmaceutical products. We therefore call these potential contaminants leachables. In biopharmaceutical production, leaching problems are most likely to occur in the bioprocessing of suspension cultures, the downstream processing of proteins, and the final packaging of liquid formulations.

Sources of L&E in biopharmaceutical production processes

Although the terms “leachables” and “extractables,” are often used in the same breath, as with the contraction, “L&E,” we need to think of them separately. Leachable compounds would typically only be present in trace quantities, so determining which compounds are present and then quantifying them analytically are very challenging—thus the expression “finding the needle in the haystack” in the title of this article. Experience has shown that it is often best to first identify the chemical species that might possibly be leached by subjecting each plastic used in the system to a rigorous process of extraction in the laboratory. Unlike leachability testing, which usually occurs under the actual production condition, storage system, or packaging, extraction tests are conducted in the laboratory without constraints—temperatures and pressures may be raised, strong acids or organic solvents added, and multiple cycles conducted—to force any possible contaminants to identify themselves. The extractables thus elicited tell us what “needles to look for in the haystack” using highly specific and sensitive analytical techniques. Substances commonly identified by extractability testing include volatiles, semi-volatiles and non-volatiles, acidic or basic compounds, inorganic elements, and conducting ions.

Common L&Es

How to deal with L&Es

Chemicals that leach into pharmaceuticals may affect the health of patients, and thus the safety of the drug. Two types of factors influence the amount and impact of L&Es in final pharmaceutical formulations: process-related and dose-related. Process factors, for example, include the contact time and contact area per volume of the pharmaceutical product with the plastic material. Process factors determine the concentration of leachables in the drug product. For drug safety, however, the leachable concentration is less important than the dosage the patient receives, and its frequency.

In a jungle of no regulations

There are no global regulatory guidelines that specify how to deal with L&Es in biopharmaceutical processing. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) only stipulate that process material should not “present any hazards to the product” and should not be “reactive, additive, or adsorptive”. There are neither binding guidelines for testing and evaluation nor any permissible limits.

Several organizations, however, are currently working together to establish best practices and guidance. The Bio-Process Systems Alliance (BPSA) works out L&E testing strategies in the English-speaking countries, while the Bioprocess Technology Working Group at DECHEMA is doing so in the German-speaking countries. [1] Both BPSA and DECHEMA recommend the implementation of L&E testing programs at an early stage of bioprocess development. The USP is expected to soon publish its new chapter <665> “Polymeric Components and Systems Used in the Manufacturing of Pharmaceutical and Biopharmaceutical Drug Products” (draft published as <661.3> [2]).

Dosage Dependance – The safety risk of L&Es in final drug formulations strongly depends on dosage and taking frequency.

Getting started with proper testing strategies

Before single-use equipment became common in biopharmaceutical production, L&Es were mainly considered for packaging. Today, most biomanufacturers use simple biological reactivity tests to evaluate the risk of L&E impurities. This is insufficient. Modern systems require comprehensive and meaningful analytics. [3]

“Searching for leachables without a prior extractable study is like looking for a needle in a haystack”, is how Desmond Hunt, the principal scientific liaison at the US Pharmacopeial Convention, was recently cited in an issues paper. [4] This quote sums up the challenges and requirements of L&E testing.

A proper testing strategy should be based on extractable data, as this provides the needed primary information on potentially leachable chemical entities. Compared to determining leachables it is quite fast and easy to search for extractables for plastic components used in a biopharmaceutical production process.

How to mitigate L&Es

The first step regarding L&Es in drug quality assurance is development of a comprehensive risk assessment strategy if there is any contact between the drug formulation and plastic components. [5] The specific risk factor for leaching should be evaluated for each material contacted. Vendor data can be a first reference here. Not only the raw material origin, but also how it is processed and pre-treated play a major role to evaluate the risk for L&Es. For example, softeners or other additives may cause leaching of components. Also, gamma ray-based sterilization of plastic materials is discussed to make polymer layers degrade which is why other methods might be preferable.Whenever a potential risk is identified, the extractable data for this contact material must be analyzed and the drug formulation needs to be tested for all possible leachable compounds.

An issue for teaming

Most experts agree that an effective testing strategy for L&Es must be based on a deep understanding of the entire process. This would need to include R&D studies, process descriptions, batch records, standard operating procedures, technical reports, batch testing, data trending, and operation parameters.

A guide to impeller selection

When growing microorganisms or mammalian cells in a stirred-tank bioreactor, it is critical to pick the impeller type that best suits the process. Selecting the wrong impeller could make chop suey of your filamentous fungi. Going for the right impeller can greatly increase yields of picky cell lines and even stem cells. With a wide variety of impeller designs how can we determine which one is best?

How blade orientation affects mixing

All stirred-tank bioreactors are designed to homogenously mix cells, nutrients, and gases. The impeller evenly distributes oxygen and substrates to the cells for healthy growth. It keeps them from settling to the bottom of the vessel and helps maintain a stable, even temperature. Depending on the impeller you choose, mixing will be achieved by radial or axial flow – or a combination of both.

Rushton and Rushton-type impellers: Ideal for fermentation

The Rushton impeller, or Rushton turbine, is the most commonly used impeller for microbial cultivation. Its blades are flat and set vertically along the agitation shaft, producing a radial flow. It is used with organisms that are relatively resistant to shear stress, such as bacteria, yeasts, and some fungi.

Pitched-blade impellers: For shear-sensitive cells

The blades on pitched-blade impellers are flat and typically oriented at a 45° angle. These impellers produce simultaneous radial and axial flows, providing efficient mass transfer. Pitched-blades are low-shear impellers that gently mix the culture without damaging the cells. They are used for mammalian and insect cell cultures growing in suspension or on microcarriers. Thanks to their proficiency in mixing, pitched-blade impellers are sometimes also used to stir viscous microbial cultures, such as filamentous fungi.

Marine impellers: Even more gentle

The leading face of the blades on a marine impeller can be flat or concave, whereas their back sides are convex. Like pitched-blade impellers, they are used in applications requiring gentle mixing. Marine impellers produce an axial flow, so their mixing efficiency tends to be slightly lower than those of impellers producing both radial and axial flows.

Packed-bed baskets: Perfect for secreted proteins

In a packed-bed basket impeller, cells are immobilized on or entrapped in a three-dimensional matrix. The basket is comprised of two horizontally positioned, perforated metal plates that extend to the vessel walls. Enclosed between the plates, Fibra-Cel disks or other matrices form a solid bed for cell growth. Cells within the bed are protected from external shear forces. A hollow impeller tube circulates medium, with discharge ports positioned above the basket. As the tube rotates it creates a small negative pressure at the bottom, drawing culture broth into the tube for recirculation. Packed-bed impellers are primarily used for manufacturing high-yields of secreted products from suspension cells or anchorage-dependent cultures in continuous cultivation.

Spin Filters: Perfusion for all cell lines

Spin filters are retention devices designed to keep the cells inside the vessel in perfusion cultivation. Combined for example with low-shear marine impellers they can be used for suspension cells and cells growing on microcarriers. The spin filter consists of a screened cage with very small pore size that keeps cells isolated outside the cage. The cage rotates, attached to the concentric impeller shaft, while inside of it a dip tube continuously withdraws the filtered, cell-free culture broth. For perfusion culture, an addition tube outside the cage provides a steady supply of fresh medium so that the working volume remains at a constant level. This mechanism is ideal for production of secreted proteins: It keeps the harvested medium free of cells, which simplifies downstream purification. Over time, however, the spin filter screen material will tend to clog and require replacement, thus limiting cultivation time.

Special impellers for microcarrier cultures

The cell-lift impeller provides ultra-low shear circulation for microcarrier cultivations. A hollow impeller tube circulates medium. As the tube rotates it creates a small negative pressure at the bottom, drawing culture broth and microcarriers up and delivering them through three discharge ports above and external to the cage. Gases are introduced through a ring sparger, generating bubbles that pass along the impeller in the so-called aeration cage. A mesh lining on the outer membrane of this cage ensures that cells growing on the microcarriers cannot pass through. Gas exchange occurs only at the membrane-media interface so that cells remain in a bubble-free environment and are not subjected to shear stress caused by bursting bubbles. The gas bubbles are then released through two ports at the top of the impeller into a second screened-in cage. Air, supplied by headspace gassing, is directed into this cage to break up foam. Cell-lift impellers are typically used in batch and fed-batch processes with shear-sensitive mammalian cells grown on microcarriers. When a decanting column and media addition tube and harvest tubes are added, they can also be used for perfusion culture.